Disposable container for bioburden sample collection and detection

Abstract

A disposable container for collection of microbe containing fluid samples is useful in a variety of microbial detection methods. One end of the container is provided with a lid comprising fluid flow apertures on one surface and a membrane filter (MF) on an opposed surface. Methods are disclosed wherein the container is disposed in a holding assembly with the lid gripped in a separate platform. The assembly and platform can be displaced relative to each other to separate the lid from the container.

Claims

1. A method of detecting microbial presence in a fluid sample, the method comprising: filling at least a portion of a collection bottle with a fluid sample containing analyte microbes therein, said collection bottle comprising opposed top and bottom openings with at least one of said openings being sealed during said filling step; providing a first cap member on one opening of said openings, said first cap member comprising a first surface comprising fluid flow apertures therein and a membrane filter (MF) layer underlying said first surface; providing a second cap member on another opening of said openings, said second cap member comprising a fluid impervious needle penetrable surface; draining said fluid sample through said first cap member thereby depositing said analyte microbes on said MF layer; injecting a reagent into said fluid sample through said fluid impervious needle penetrable surface, said reagent configured to react with said analyte microbes to form a detectable presence on said MF layer, said reagent comprising a staining reagent or a luminescent agent configured to yield luminescent microbes on said MF layer; and placing a detector in operative association with said MF layer to detect said analyte microbes.

2. The method as recited in claim 1, wherein said detector is a microscope.

3. The method as recited in claim 1, wherein said detector comprises a camera.

4. The method as recited in claim 1, further comprising placing said collection bottle in a bottle holding assembly, and after said step of injecting said reagent into said fluid sample, separating said first cap member from said collection bottle.

5. The method as recited in claim 4, further comprising securing said first cap member in a gripping member carried by a platform, wherein said platform and said bottle holding assembly are displaced relative to each other to thereby separate said first cap member from said collection bottle.

6. The method recited in claim 1, further comprising connecting a fluid conduit to said first cap member, and draining said fluid sample through said fluid conduit.

7. The method as recited in claim 6, further comprising applying a vacuum source to said fluid conduit.

8. A method of detecting microbial presence in a fluid sample, the method comprising: filling at least a portion of a collection bottle with a fluid sample containing analyte microbes therein, said collection bottle comprising opposed top and bottom openings with at least one of said openings being sealed during said filling step; providing a first cap member on one opening of said openings, said first cap member comprising a first surface comprising fluid flow apertures therein and a membrane filter (MF) layer underlying said first surface; providing a second cap member on another opening of said openings, said second cap member comprising a fluid impervious needle penetrable surface; draining said fluid sample through said first cap member thereby depositing said analyte microbes on said MF layer; and placing a detector in operative association with said MF layer to detect said analyte microbes.

9. The method as recited in claim 8, wherein said detector is a microscope.

10. The method as recited in claim 8, wherein said detector comprises a camera.

11. The method as recited in claim 8 further comprising placing said collection bottle in a bottle holding assembly, and separating said first cap member from said collection bottle.

12. The method as recited in claim 11, further comprising securing said first cap member in a gripping member carried by a platform, wherein said platform and said bottle holding assembly are displaced relative to each other to thereby separate said first cap member from said collection bottle.

13. The method recited in claim 8, further comprising connecting a fluid conduit to said first cap member, and draining said fluid sample through said fluid conduit.

14. The method as recited in claim 13, further comprising applying a vacuum source to said fluid conduit.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a side elevation view of a bioburden sample selection and detection container with certain parts shown in phantom;

(2) FIG. 2 is a side elevation view of the container of FIG. 1 with the protective film removed from the top cap;

(3) FIG. 3 is an exploded side view of the container with certain parts of the container removed;

(4) FIG. 4 is a partially cut-away orthogonal projectional view of the container, associated holding assembly and platform during the drainage stage in which fluid is evacuated from the container; and

(5) FIG. 5 is a partially cut-away view similar to FIG. 4 but showing another stage in the bioburden testing process in which the platform carrying the membrane filter (MF) is separated from the container holding assembly.

DETAILED DESCRIPTION

(6) Turning to FIGS. 1-3, there is shown a disposable container 2 for bioburden sample selection and detection. The container is composed of a clear plastic material such as polycarbonate, polyethyleneterephlate, polypropylene, or other plastic material. As shown, the container comprises cylindrically cross sectioned body section 8 and a tapered or decreasing diameter section 10 in the general form of a truncated conical shape superposed over the body 8. As shown in FIG. 1, the top most section of the container is defined by a narrowed neck portion 9 over which the cap sections are adapted for engagement by threaded engagement, snap fit, or other like attachment means. With specific reference to FIG. 1, a protective film 14 is provided on the top of the container and is, for example, shrink fit over the top of the container for easy removal. The top cap 12 also includes an annular sealing lid portion 17. The top cap assembly fits over and seals top opening 5 of the container.

(7) At the bottom end of container 2, a removable cap 20 is provided over the bottom or base of the bottle. A fluid impervious rubber diaphragm 26 or similar surface is provided along the inside lower extremity of the bottle, and this may be secured to the bottom of the bottle via sealing cap 24 that can be threaded or snap fit over the bottle base. The cap 20 seals the bottom opening 7 of the container.

(8) In FIG. 2, the protective film 14 has been removed from the top cap 12 revealing annular sealing lid 17 that, in turn, is more particularly friction fit within the circumference of the neck 9. Annular sealing lid 17 comprises a raised annular land area surrounding a recessed, planar section having a multiplicity of apertures 16 therein to facilitate fluid flow through sealing lid 17. On the underneath or bottom side of lid 17 is a filtration membrane 18 that may be composed of polyester, polycarbonate, polypropylene, polytetrafluorethylene, nylon, and poly(vinylidene difluoride). This membrane functions to retain the microbial species thereon as shall be referred to in detail later.

(9) FIG. 3 shows the container in inverted disposition. This is the orientation in which, in one embodiment, the bottle will be positioned in order to facilitate bioburden sampling and detection. Here, removable outer cap 20 has been removed, revealing the annular securing cap 24 and underlying fluid impenetrable rubber layer 26. The layer 26 is adapted to act as a barrier to fluid flow therethrough, but this layer may be penetrated by a sharp object such as a hypodermic needle or the like. More particularly, layer 26 is formed of an elastomeric material such as EPDM rubber, styrene butadiene, acrylonitrile/butadiene, polychloroprene, or natural rubber. Also, in FIG. 3, the protective film 14 has been removed from the now inverted top of the bottle, revealing filtration membrane 18 on the bottom side of annular snap lid 17 with the recessed aperture side 16 of lid now disposed under the filtration membrane 18.

(10) In order to perform bioburden testing, the bottle 2 is filled with aqueous or other fluid sample containing the analyte microbial species. The bottle 2 may be placed in a bottle holding assembly 100 such as the type shown in FIG. 4. Here, assembly 100 comprises two opposed platen members 102, 104, with the top platen 102 joined to platen 104 via connectors 106, 108, 110, 112 which can all be adjusted through the spring tension supplied by coil spring 114 and 116. A retainer member 120 such as composed of a rubber diaphragm or similar resilient material depends downwardly from the platen 102 and has a general shape that is congruent to the shape of bottle 2 to securely grasp and hold same in the assembly 100.

(11) As shown in FIG. 4, platform 140 is positioned underneath the platen 104 and the annular sealing lid 17 is engaged between yoke members 142, 144 carried on the platform, which together, encircle the lid 17. Set screws or the like may be used to vary the tension of springs 146, 148 to provide for secure grasping of lid 17 by the yoke members. At this point, the protective film 14 has been removed from the tap 12, so that the membrane 18 (FIG. 3) is in direct fluid communication with the contents of the bottle 2. A conduit 150 is connected via a funneling type connector or other device (not shown) to the now inverted top section of the bottle. A vacuum source 154 or pump communicates with the bottle 2 via the provision of valve member 152. A hypodermic rubber needle or the like is inserted through the penetratable rubber layer of the bottle to inject staining, luminescence, cleaning, or other reagent or purified water to the fluid sample within bottle 2.

(12) By turning on the vacuum or pump, the fluid in the sample is drained out through the filtration membrane 18 and the draining aperture 16 on the lid 17 to waste. Due to the pore size of the filter membrane, the microbial species of the sample will be captured on the filtration membrane 18, and then the pump or vacuum source is turned off. Thus, after draining the fluid from the bottle, the microbial species and reagent are disposed on the membrane 18 so that the microbial count may then be made via conventional techniques.

(13) Turning to FIG. 5, as shown, the platform 140 has been removed from the lower platen 104 with the microbial species and reagent disposed on the surface 18. Immediately prior to this step, the lid 17 has been removed from the remainder of the container, leaving lid 17 disposed on the platform. As shown in FIG. 5, the spring tension in springs 146 and 148 has been relaxed with the lid 17 loosely encircled by the yoke members 142, 144. At this stage, a detector such as a microscope or the like can be moved close to the membrane to count or otherwise detect the analyte microbial species. After the inspection, the bottle 2 may be disposed of.

(14) It is thus apparent that a disposable container for bioburden sample selection and detection is provided. This container works as a normal bottle to select the fluid sample and is sealed by a cap. In one aspect of the invention, the container is then placed upside down on stages for sample preparation (filtration, staining) and detection (imaging).

(15) The bioburden measurement process may involve any one of many conventional techniques such as a colormetric measurement technique in which a staining reagent is injected into the fluid sample in container 2 via a syringe or the like that penetrates the layer 26. A microscope or the like is used to count the microbial colonies on the membrane 18 to assess bioburden level.

(16) Additionally, another exemplary bioburden test method may comprise injection of a luminescent or fluorescent reagent into the sample in the container 2 through the penetrable layer 24. The desired analyte microbial species captured on the surface of membrane 18 can then be detected via a camera or the like placed proximate the membrane 18.

(17) Another bioburden testing procedures that may be utilized involves the “in situ cell division” method. This method is a direct growth rapid method for living target cell detection before the cells become visible to the naked eye. In this method, the sample is filtered through the MF to capture the microorganism thereon. The MF is transferred to a nutrient agar plate and the target cells are allowed to form microcolonies by in situ respiration. (This usually takes about a three-hour incubation time.) The microcolonies are illuminated with a blue light LED (Ex 450-500 nm, Em 510-560 nm). A CCD chip is used to detect cellular autofluorescence and analysis software is used to automatically count the photosensitive pixels overlying each microcolony.

(18) Another exemplary bioburden test procedure is known as the “solid phase cytometry (SPC) method”. This method is a rapid method for detection of total viable cells (TVC) by the sequential steps of: (a) filtering a sample over a black polyester or polycarbonate MF to capture microorganisms; (b) transferring the MF to a pad comprising fluorocarbon dye, incubating, to label metabolically active cells only; (c) detecting fluorescence emitted by labeled cells with argon laser scanning (Ex 480 nm, Em 515 nm); (d) processing signals by computer to differentiate valid signals from fluorescent particles; and (e) quantifying total viable cells.

(19) The pore size of the filter membrane is chosen to capture the secured microbial analyte thereon. This pore size can vary over a wide range such as to encompass pore sizes from about 0.05 μm to about 0.65 μm. Exemplary membrane filters (MF) include the following: 1) 25 mm diameter, 7-20 μm thickness, 0.4 μm pore size; 2) 25 mm diameter, 6-11 μm thickness, 0.2 μm pore size; and 3) 47 mm diameter, 7-22 μm thickness, 0.45 μm pore size. The selection of the appropriate filter membrane 18 is within the skill of the artisan.

(20) The artisan will appreciate that bioburden testing can be conducted to determine presence and count levels of a variety of microbes. For example, counts of a variety of bacteria, fungi, mold, yeast, and spores can be conducted using the container and methods of the present invention.

(21) It is to be understood that the invention is not limited to the illustrations described and shown herein, which are deemed to be merely illustrative of various embodiments of the invention and which are suitable for a variety of modifications. The invention is intended to encompass all such modifications which are in the spirit and scope as defined in the attached claims.